Stable, homogeneous natural product extracts containing polar and apolar fractions

ABSTRACT

Disclosed are stable, homogeneous dispersions, comprising (a) from about 20 to 90% by weight of a first composition comprising (i) about 60-95% by weight of a first polar solvent, (ii) about 0-40% by weight of one or more second polar solvents, and (iii) water soluble components of a first natural product; and (b) from about 10 to 60% by weight of a second composition comprising: (i) one or more apolar solvents, and (ii) oil soluble organic components of a second natural product; and (c) from about 0.01 to 8% by weight of a non-surface active lipid phosphate or a surface active agent. Also disclosed are methods of forming the stable, homogeneous dispersions of the invention, comprising forming a first composition comprising water soluble components of a first natural product; forming a second composition comprising oil soluble organic components of a second natural product; mixing the compositions and subjecting the mixture to high pressure high shear processing to form a stable, homogeneous dispersion.

[0001] This application is a divisional of U.S. patent application Ser.No. 09/544,649, filed Apr. 6, 2000, which claims priority under 35U.S.C. § 119(e) of provisional application serial No. 60/127,930, filedApr. 6, 1999.

FIELD OF THE INVENTION

[0002] This invention is directed to the field of compositionscomprising natural product extracts for use in personal care products orpharmaceuticals.

BACKGROUND OF THE INVENTION

[0003] Natural products and natural product extracts are often used incosmetic and pharmaceutical applications. Natural products, particularlybotanically sourced natural products, have demonstrable beneficialproperties on the skin and hair. Extracts of these natural products havedemonstrated antimicrobial, antiseptic, anti-inflammatory, antioxidant,enzyme stimulation or inhibition, pigmentation enhancement or control,photoprotective and many other physiological benefits.

[0004] Typically, the solvent systems used to produce these extracts arepolar in nature, and most typically are water or glycols or acombination thereof. These polar solvents enable the extraction of onlya similarly polar material from the biomass of the natural productmaterial. Similarly, apolar solvents have been used to remove the apolarfractions from the biomass of the natural product materials. There iscurrently no single universal solvent capable of only extracting boththe desired polar and apolar fractions. As a result, typical naturalproduct extracts only provide a portion of the physiologically oraesthetically beneficial components.

[0005] It has not been possible to date to combine an aqueous extractwith a lipid or other apolar phase into a single system without the useof surface active agents and special processing conditions to formemulsions or dispersions. Surface active agents permit the mixing of ahydrophilic phase and a hydrophobic phase by lowering the surfacetension between the two phases, thereby creating micellar structureswhich, when mixed with a suitable processing procedure, produce stablesystems. However, these surface active agents may be irritating tousers, and the processes used to prepare the emulsions or dispersionmake them difficult to reproduce.

[0006] Since many bioreactive or aesthetic components of the naturalproduct are located within the structure of the cell wall or otherorganelles within the cell, a suitable process is required to extractthe desired components from the cell. Simple solvent extraction isusually insufficient to remove the protected material from the cell. Thecell wall barrier must be perturbed or ruptured sufficiently to allowdiffusion to occur into the extracting solvent. A method is thereforeneeded to rupture cell walls and membranes to maximize the removal ofthe physiologically active or aesthetically pleasing materials. Examplesof such process conditions include the use of heat, high shear mixing,ultrasonic waves, microwaves, high pressure and prolonged polar orapolar dialysis.

[0007] It is preferred to combine the process described above with theuse of separate polar and apolar solvents to remove the maximum amountof all of the materials of interest. It is further preferred to combineboth the polar and apolar extracts into a single, homogeneouspreparation without the use of surface active agents which can causeirritation.

OBJECTS OF THE INVENTION

[0008] It is therefore an object of the present invention to provide animproved method of obtaining a composition comprising both thehydrophobic and hydrophilic components of a natural product or blend ofnatural products.

[0009] It is another object of the present invention to provide a singlecomposition having both oil soluble and water soluble components of anatural product extract, in order to take advantage of synergy which isoften associated with the use of multiple components of a naturalproduct.

[0010] It is another object of the present invention to provide agreater functional activity due to the presence of a full complement ofpolar and apolar materials with physiologically beneficial oraesthetically pleasing properties.

[0011] It is another object of the present invention to provide acomposition having greater potency and stability of the desiredcomponents, in comparison to prior art compositions.

[0012] It is another object of the present invention to provide betterdelivery of the physiologically beneficial active agents to sites withinthe skin or hair, where the active agents are needed for activity.

[0013] It is another object of the present invention to provide ease ofmanufacture of a finished system containing the full complement ofextracts, yielding cost savings and greater reproducibility withconsistent quality standards.

[0014] It is another object of the present invention to provide agreater versatility to compositions of natural product extracts that canbe achieved by preparing prior art homogeneous or heterogeneouscomplexes.

[0015] It is another object of the present invention to provide acomposition useful for personal care, pharmaceutical or cosmeticapplications, having low irritation due to the absence of surface-activeagents that lower the surface tension between immiscible phases.

[0016] Applicants have discovered methods of forming stable, homogeneouscompositions comprising both polar and apolar fractions of naturalproduct extracts. The applicants have now discovered that aqueous oraqueous/glycolic extracts can be successfully mixed with apolar extracts(e.g. silicone extracts) using high pressure, high shear processing toproduce a single, stable homogeneous system that contains the entirecomplement of polar and apolar fractions from a selected naturalproduct.

SUMMARY OF THE INVENTION

[0017] The invention is directed to stable, homogeneous dispersionscomprising a water-soluble natural product extract and an oil solublenatural product extract. In certain embodiments, the dispersion does notcomprise a surface active agent.

[0018] In one embodiment, the invention is directed to stable,homogeneous dispersions comprising a first composition which containsone or more polar solvents and water (or polar solvent) solublecomponents of a first natural product; and a second compositioncomprising one or more apolar solvents and oil (or apolar solvent)soluble organic components of a second natural product. Optionally, thedispersion may also comprise from about 0.01 to 8% by weight of anon-surface active lipid phosphate or a surface active agent.

[0019] In preferred embodiments, the invention is directed to stable,homogeneous dispersions comprising

[0020] (a) from about 20 to 90% by weight of a first compositioncomprising:

[0021] (i) about 60-95% by weight of a first polar solvent;

[0022] (ii) about 0-40% by weight of one or more second polar solvents;and

[0023] (iii) water soluble components of a first natural product; and

[0024] (b) from about 10 to 60% by weight of a second compositioncomprising:

[0025] (i) one or more apolar solvents; and

[0026] (ii) oil soluble organic components of a second natural product;and

[0027] (c) from about 0.01 to 8% by weight of a non-surface active lipidphosphate or a surface active agent.

[0028] In particular embodiments, the first polar solvent may be waterand the second polar solvent is selected from the group consisting ofwater; a mono, di, tri or polyhydroxy alkyl derivative; a mono, di, trior polyhalogenated alkyl derivative; a mono, di, tri or poly alkyl etherderivative; and a mono, di, tri or poly carboxyl alkyl derivative. Theapolar solvent may be one or more of an oil (such as a vegetable oil); amono, di, tri or polyalkyl ester or ether of a mono, di, tri orpolyhydroxy compound; a saturated, unsaturated, linear, branched, orcyclic hydrocarbon; a saturated, unsaturated, linear or branched C₈ toC₃₀ fatty acid; a branched, linear, or cyclical silicone or siliconederivative; or a homopolymer or heteropolymer fluid formed by thepolymerization of alkylene oxide monomers.

[0029] The dispersion may comprise components from any of the naturalproducts which are known to contain physiological properties. Examplarynatural products are mulberry, lavender, licorice root, arnica,eyebright and grape root. The invention contemplates the use of waterand oil soluble components of natural products which may be obtained byany of the methods known to those skilled in the art.

[0030] The invention is also directed to methods of forming thedispersions of the invention. In one embodiment, the process includesthe steps of obtaining a first natural product extract comprising apolar solvent and water based components of a natural product; obtaininga second natural product extract comprising an apolar solvent and oilsoluble components of a natural product; combining the first and secondextracts to form a composition; optionally adding about 0.01 to 8% byweight of a non-surface active lipid phosphate or a surface activeagent; and subjecting said mixture to high pressure high shearprocessing to form a stable, homogeneous dispersion.

[0031] Preferred rates of high pressure, high shear processing are at apressure of about 11,000 to about 27,000 psi, and at a shear rate whichis sufficient to form a dispersion having an average particle size offrom about 200 to about 1,000 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a chromatogram of a composition containing the polarsolvent soluble components of mulberry;

[0033]FIG. 2 is a chromatogram of a composition containing the apolarsolvent soluble components of mulberry;

[0034]FIG. 3 depicts the tyrosinase inhibitory activity of Example 15,comprising both the polar and apolar solvent soluble organic componentsof mulberry;

[0035]FIG. 4 is a chromatogram of a composition containing the apolarsolvent soluble components of licorice;

[0036]FIG. 5 is a chromatogram of a composition containing the polarsolvent soluble components of licorice;

[0037]FIG. 6 depicts the tyrosinase inhibitory activity of Example 17,comprising both the polar and apolar solvent soluble organic componentsof licorice;

[0038]FIG. 7 depicts the melanocyte pigmentation reduction of Example17, comprising both the polar and apolar solvent soluble organiccomponents of licorice;

[0039]FIG. 8 is a chromatogram of a composition containing the polarsolvent soluble components of lavender;

[0040]FIG. 9 is a chromatogram of a composition containing the apolarsolvent soluble components of lavender;

[0041]FIG. 10 depicts the results of a neutrophil elastase inhibitionassay of Example 16, containing both the polar and apolar solventsoluble organic components of lavender, and a comparison with the assayresults for conventional prostaglandin;

[0042]FIG. 11 depicts the results of inhibition of UV-induced PGE₂release of Example 18, containing both the polar and apolar solventsoluble organic components of arnica;

[0043]FIG. 12 is a chromatogram a composition containing the polarsolvent soluble components of eyebright;

[0044]FIG. 13 is a chromatogram of a composition containing the apolarsolvent soluble components of eyebright;

[0045]FIG. 14 depicts the results of a cytochrome C reduction assay ofExample 19, containing both the polar and apolar solvent soluble organiccomponents of eyebright; and

[0046]FIG. 15 depicts the results of a cytochrome C reduction assay ofExample 20, containing both the polar and apolar solvent soluble organiccomponents of grape root.

DETAILED DESCRIPTION OF THE INVENTION

[0047] All patents, applications, test methods and publicationsreferenced in this specification are hereby incorporated by reference intheir entirety. In case of conflict, the present description, includingdefinitions, will prevail.

[0048] As used herein, the terms “water soluble” and “polar solventsoluble” are used interchangeably, and refer to the property of beingsoluble at approximate room temperature and one atmosphere pressure inwater or another polar solvent commonly used in preparing cosmeticformulations, such as a glycol.

[0049] As used herein, the term “polar solvent soluble components”refers to the components of a natural product which may be extractedfrom the natural product by contacting the natural product with a polarsolvent, for example water or glycol or mixtures thereof.

[0050] As used herein, the terms “oil soluble” and “apolar solventsoluble” are used interchangeably, and refer to the property of beingsoluble at approximate room temperature and one atmosphere pressure inan oil or other apolar solvent commonly used in preparing cosmeticformulations, such as silicone based solvents or oils or mixturesthereof.

[0051] As used herein, the term “organic oil soluble components” refersto the components of a natural product which may be extracted from thenatural product by contacting the natural product with an apolarsolvent, such as silicone or oils or mixtures thereof.

[0052] As used herein, the term “extracts” or “natural product extracts”are used interchangeably, and refer to the organic components of anatural product which can be obtained by contacting the natural productwith a polar or apolar solvent.

[0053] As used herein the term “surface-active” or “surface-activeagent” refers to a substance capable of reducing the surface tension ofa liquid in which it is dissolved. A “non-surface active agent” is asubstance which does not effectively reduce the surface tension of aliquid in which it is dissolved.

[0054] As used herein, the term “surfactant” refers to a surface-activesubstance.

[0055] As used herein, the term “surfactant-free dispersion” refers to astable or dispersion that is produced without the use of surface-activeingredients or surfactants.

[0056] The present invention is directed in part to compositionscontaining natural product extracts which can be used in personal careand pharmaceutical formulations and products. In some embodiments, theinvention is directed to a natural product extracts or combinations ofnatural product extracts containing hydrophilic and hydrophobicmaterials which are processed to produce a composition withphysiologically beneficial and aesthetically pleasing properties. Thesenatural products can be either animal or vegetable derived. In preferredembodiments, the invention is directed to compositions comprisingnatural product extracts from land-based or marine-derived botanicals.

[0057] In one embodiment, the compositions of this invention aredispersions which may comprise both polar solvent soluble components ofa natural product and apolar solvent soluble components of a naturalproduct. The polar solvent soluble components may be present in acomposition also comprising one or more polar solvents, and the polarsolvent soluble components may be present in a second composition, alsocomprising one or more apolar solvents.

[0058] The first composition used to form the dispersion may contain theorganic polar solvent soluble components of a natural product. Inpreferred embodiments, the dispersion may comprise a first composition,which contains polar solvent soluble components of a natural product.The first composition may contain first and second polar solvents. Thefirst polar solvent may be present in the amount of about 60 to 95% byweight of the first composition. The first composition may also comprisea second polar solvent, which may be present in the amount of about 0 to40% by weight.

[0059] In preferred embodiments, the first polar solvent is water andthe second polar solvent is a glycol, such as propylene glycol orethylene glycol.

[0060] The first composition may be present in the amount of about 20 to90% by weight of the dispersion, and is preferably present in the amountof 40 to 80 wgt %, more preferably 50 to 70 wgt %, most preferably 60 to65 wgt %.

[0061] The dispersion also comprises the organic apolar solvent solublecomponents of a natural product, for example in the form of a secondcomposition which contains apolar solvent soluble organic components ofa natural product and an apolar solvent.

[0062] The second composition maybe present in the amount of about 10 to60% by weight of the dispersion, and is preferably present in the amountof 20 to 50 wgt %, more preferably 30 to 40 wgt %, and most preferablyabout 30 wgt %.

[0063] In certain embodiments, the natural product extracted in thefirst and second composition are the same. However, the invention alsocontemplates dispersions in which the natural product extracts presentin the first and second compositions are obtained (or extracted) fromdifferent natural products. In addition, the invention contemplatesdispersions in which either (or both) of the first and secondcompositions comprises extracts or components of more than one naturalproduct.

[0064] In preferred embodiments, the dispersion is a surfactant freedispersion, and does not contain a surface active agent.

[0065] Phospholipids are one agent which may be used to achievesurfactant-free dispersions of the invention. Phospholipids in generalare not soluble in water. In contrast, surface active materials reducethe surface tension of the liquid in which it is dissolved.

[0066] For example, when lecithin is refined and the concentrations ofphosphatidyl choline increase, the surface tension of dispersions of thephospholipid in water increase. At concentrations of 50% phosphatidylcholine and higher, the surface tension exceeds the surface tension ofwater alone by a very considerable level. The conditions involved forthe surface tension measurement are for a condensed, solid monolayer ofphosphatidyl choline. The ability to achieve the monolayer is dependenton first processing the phosphatidyl choline above its phase transitiontemperature of 41° C., and then allowing the condensed monolayer tocool. This notion of phase transition associated with increased energyis important in consideration of phospholipids, and particularlyphosphatidyl choline, as a means of achieving surfactant-free emulsions.

[0067] In particular embodiments, the dispersion may also comprise from0.01% to 8% by weight (preferably 0.01 to 5% by weight) of one or morelipids. Examplary lipids include Phospholipon 80, 80H (AmericanLecithin) and Basis LP2OH (Ikeda Corp., Japan).

[0068] The components of the natural products may be obtained by variousmethods known to those of ordinary skill in the art. Polar solventsoluble components of a natural product may be obtained by contactingthe natural product with a polar solvent to form a solution containingthe polar solvent soluble components, optionally mixing the solution andoptionally thereafter diluting the solution with another polar solvent.For example, a natural product can be contacted with water to form asolution containing water soluble components of the natural product, thesolution may be mixed, and thereafter may be diluted with glycol.Alternatively, the natural product can be contacted with glycol to forma solution containing organic water soluble components of the naturalproduct, the solution may be mixed, and thereafter diluting the solutionwith water. In still another method, the natural product can becontacted with both water and glycol to form a solution, and optionallymixed.

[0069] Polar solvent soluble (or water soluble) components of a naturalproduct are obtained by contacting the natural product with a polarsolvent, for example water, glycol, or mixtures thereof, to form asolution containing the polar solvent soluble components. If necessary,the solution may thereafter be mixed or stirred until the solution isclear.

[0070] Suitable polar solvents include water; glycols; mono, di, tri orpolyhydroxy alkyl derivatives; mono, di, tri or polyhalogenated alkylderivatives; mono, di, tri or polyalkyl ether derivatives; and mono, di,tri or polycarboxyl acid derivatives and mixtures thereof. Exemplaryglycols include ethylene glycol, propylene glycol, 1,3-butylene glycoland glycerin.

[0071] Organic apolar solvent soluble (or oil soluble) components of anatural product are obtained by contacting the natural product with anapolar solvent, for example cyclomethicone, hydrogenated polyisobutene,or combinations thereof, to form a solution containing the organic polarsolvent soluble components. If necessary, the solution may thereafter bemixed or stirred until the solution is clear.

[0072] Suitbable apolar solvents include mono, di, tri or poly alkylester or ether of a di, tri, or polyhydroxy compound, such as ethyleneglycol, propylene glycol, glycerin, sorbitol or other polyol compound.Examples of such esters and ethers include, but are not limited to,saturated and unsaturated, linear and branched vegetable oils, such assoybean oil, babassu oil, castor oil, cottonseed oil, chinese tallowoil, crambe oil, perilla oil, danish rapeseed oil, rice bran oil, palmoil, palm kernel oil, olive oil, linseed oil, coconut oil, sunfloweroil, safflower oil, peanut oil and corn oil. Preferred saturated andunsaturated vegetable oils are those having fatty acid components with 6to 24 carbon atoms. A more preferred vegetable oil is soybean oil.

[0073] Additional exemplary apolar solvents include compounds having theformula C_(n)H_((2n+2−m)) where n is an integer greater than or equal to6 and m is 0 or an even integer no greater than n. Such compoundsinclude, but are not limited to, saturated and unsaturated, linear,branched, and cyclic hydrocarbon chains. Preferred examples of suchcompounds include, but are not limited, mineral oil, petrolatum,permethyl fluids, polybutylenes, and polyisobutylenes.

[0074] Further apolar solvents contemplated for use in the inventionhave the formula

[0075] or the formula

[0076] where R₁ is a saturated or unsaturated, linear, branched orcyclic C₁-C₂₄ alkyl; R₂ is hydrogen or a saturated or unsaturated,liner, branched or cyclic C₁-C₂₄ alkyl; and n is an integer from 0 to20. Examples of such aesthetic modifying agents include, but are notlimited to, isopropyl palmitate and diisopropyl adipate.

[0077] Yet another group of apolar solvents is silicone and siliconederivatives. Silicone may provide lubrication and/or shine to thecomposition. Preferably, the silicone is insoluble in water. Suitablewater-insoluble silicone materials include, but are not limited to,polyalkylsiloxanes, polyarylsiloxanes, polyalkylarylsiloxanes,polysiloxane gums and polyethersiloxane copolymers. Examples of suitablesilicone materials are disclosed in U.S. Pat. Nos. 4,788,006; 4,341,799;4,152,416; 3,964,500; 3,208,911; 4,364,837 and 4,465,619, all of whichare incorporated herein by reference.

[0078] Another suitable hydrophobic material which can be suspended inthe composition has the formula

[0079] where R₁ is a saturated or unsaturated, linear, branched orcyclic alkyl having 2 to 24 carbon atoms; M⁽⁺⁾ is N⁺R₂R₃R₄R₅; R₂, R₃ andR₄ are hydrogen or a saturated or unsaturated, linear or branched alkylor hydroxyalkyl having from 1 to 10 carbon atoms; and R₄ is a saturatedor unsaturated, linear, branched or cyclic alkyl or substituted alkylhaving 2 to 24 carbon atoms. An example of such a material is lauramineoleate.

[0080] Another apolar solvent is a polymer formed by polymerization ofalkylene oxide monomers of the formula

[0081] where n is from about 2 to about 24. The polymer may be either ahomogenous polymer or a copolymer. Examples of such homogenous polymersinclude, but are not limited to, polypropylene oxide and polybutyleneoxide. Generally, the molecular weight of these polymers ranges fromabout 100 to about 10,000 daltons. Additionally, these polymers may bereacted with mono or polyhydroxyalkyl alcohol, such as UCON fluids fromthe Union Carbide Chemical Company, or with a saturated or unsaturated,linear, branched or cyclic C₁-C₂₄ alkyl.

[0082] Suitable apolar solvents include oils (both natural andsynthetic), including hydrogenated oils. Exemplary oils includevegetable oil, soybean oil, babasu oil, castor oil, cottonseed oil,grapeseed oil, rice bran oil, canola oil, palm oil, palm kernel oil,olive oil, linseed oil, coconut oil, sunflower oil, safflower oil,peanut oil, corn oil, mineral oil and petrolatum. Other apolar solventscontemplated for use in the invention include hydrogenatedpolyisobutene, permethyl fluids, polyisobutene, polybutene,polypropylene oxide, polybutylene oxide, isopropyl palmitate, disopropyladipate or mixtures thereof.

[0083] Exemplary silicone and silicone derivatives include branched orlinear cyclical silicone or silicone derivatives, cyclomethicone,dimethicone polysiloxane, dimethicinol, polysiloxanes, polysiloxanecopolymers, polyalkyl aryl silanes, polyaryl siloxanes, polyalkylsiloxanes, polyalkyl aryl silanes, polysiloxane copolymers, lowviscosity dimethicone, phenyl trimethicone (Dow Corning), silicone fluidDC 345 (Dow Corning), polysynlane (NOF Corp.).

[0084] Still more suitable apolar solvents include mono, di, tri orpolyalkyl esters or ethers or a mono, di, tri or polyhydroxy compound;saturated or unsaturated, linear or branched C₈-C₃₀ fatty acids; andhomopolymer or heteropolymer fluids formed by the polymerization ofalkylene oxide monomers.

[0085] The dispersions of the invention may be produced by mixing awater soluble natural product extract and an oil soluble natural productextract, using high pressure/high shear conditions to produce ahomogeneous, fluid dispersion which is stable for a commerciallyrelevant period of time, e.g. between about 180 to 720 days when storedat approximate room temperature, in a commercial package. The preferredpressure for preparation of this dispersion is between about 11,000 toabout 27,000 psi, more preferably 11,000 to about 21,000 psi, mostpreferably between about 11,000 and 16,000 psi. The composition may beproduced with a shear that creates average particle size of betweenabout 100 to about 1,000 nm, more preferably between about 100-500 nm.

[0086] The invention is also directed in part to processes forefficiently rupturing the cell wall or lipid bilayer membrane ofindividual cells or subcellular organelles of the natural product tomaximize the removal of the cellular components.

[0087] In certain embodiments, composition of the invention may comprisenon-surface active lipid phosphate phospholipids, preservatives such asGermazide™ MPB, and nonionic detergents such as polyoxethylene ethers.The dispersion may also comprise polyethylene glycol and butylene glycolto improve the freeze thaw stability of the preparations.

[0088] Exemplary non-ionic detergents include polyoxylated ethers suchas Brij detergents, available from Sigma Aldrich Chemical Co.

[0089] Suitable natural products contemplated for use in the inventioninclude any of the natural products which are know in the art to containcomponents having physiological attributes. Exemplary natural productsinclude mulberry (morus alba), lavender (lavandula angustifolia),licorice root (glycyrrhiza glabra), arnica (arnica montana), eyebright(euphrasia officinalisa), grape root (mahonia aquifolum), green tealeaves (camelia sinesis), rosemary powder, echinacea herb powder,evening primrose flowers, sea parsley powder (palmaria palmata),calendula leaves and tea tree leaves.

[0090] Recent research has shown that mulberry (morus alba) and licorice(glycyrrhiza glabra) root extracts act as tyrosinase inhibitors.Chemical analysis has shown that the plant contains a number ofinteresting biochemicals, in particular oxyresveratrol, umbelliferone,kowano-A, kowano-B, kowano-C, kowano-F, kowano-G, kowano-H,chalcomoracin, cyclomorusin, cyclomulberrin, cyclomulberrochromenemoracenin-D, morusin, mulberranol, mulberrochromene, mulberrofuran-B,oxydihydromorusin, sitosterol and sitosterol-alpha-glucoside. Thechemical or chemical(s) responsible for enzyme inhibition has not beenunequivocally identified however researchers have shown thatoxyresveratrol is a very potent tyrosinase inhibitor (Shin et al.,Oxyresveratrol as the Potent inhibitor on Dopa Oxidase Activity of aMushroom Tyrosinase, BBRC, 1998, vol. 243, pp 801-803).

[0091] Licorice root extract is used traditionally in balms for itspowerful anti-inflammatory, anti-allergic and anti toxin properties.Recently, researchers have discovered that licorice root extractinhibits enzyme activities, especially tyrosinase and11-beta-hydroxysteroid dehydrogenase and as a result it causes skinwhitening and potentates the action of hydrocortisone. It is thereforeof great interest to the pharmaceutical and cosmetic industries.

[0092] The major component of licorice root, glycyrrhizic acid, isimportant to the food industry. It is 50 times sweeter than sucrose (A.Chevallier, The Encyclopedia of Medical Plants p 99, 1996) making it avery useful food ingredient. Other active components of Licorice includethe triterpine glycosides, glycyrrhizin, flavonoids, isoflanonoids,kumatakenin; licoricone, glabrol, glabrone, glyzarin, licoisoflavones Aand B, glycyrol, formononetin, liquiritigenin, liquiritin,neoliguiritin, rhamnoliquiritin, glyzaglabin,7-hydroxy-2-methylisoflavone, 4,7-dihydroxyflavone, glabranine,chalcones, coumarins, triterpenoids, sterols (including betasitosterol,stigmasterol), amino acids, gums, wax and volatile oil.

[0093] Lavender is associated with youthfulness. It is carminative,antiseptic and soothing. It is known amongst herbalists as a holisticrelaxant or as “the balancing” herb. Lavender extracts have beenanalyzed and the major components, linalool and linalyl acetate, arejust two among of the hundreds of different active compounds identified.Other significant components include triterpenes (e.g. ursolic),flavonoids (e.g. luteolin) and coumarins (A. Y. Leung et al.,Encyclopedia of Common Ingredients Used in Foods and Cosmetics, 1996, pp339-342). The ursolic acid and other components are known to beanti-inflammatory. The cosmeceutical benefits claimed for ursolic acidinclude restoration of overall health and functionality of photoagedskin, in addition to facilitation of tissue repair. Ursolic acid is apotent elastase inhibitor (Q. Ying et al., “Inhibition of HumanLeukocyte Elastase by Ursolic Acid; Evidence for a Hydrophobic BindingSite for Pentacyclic Triterpenes, 1991, Biochem. J. 277, 521-526). Skinis a very dynamic tissue with degradation taking place along side repairand renewal. Compositions containing lavender extract improve skinintegrity by slowing elastin degradation with respect to natural ongoingsynthesis.

[0094] The mechanism by which lavender extracts ‘relax’ and reducemuscle tension has been studied in depth. M. Lis-Balchin et al.,“Studies on the Mode of Action of the Essential Oil of Lavender(Lavender Angustifolia)”, Phytother. Res., 1999, 13(6), 540-542), showedthat muscle tension is reduced through a postsynaptic action and not viaan atropine-like mechanism and that the action of linalool reflectedthat of the whole lavender oil. (H.. M. Kim et al., “Lavender OilInhibits Immediate Type Allergic Reaction in Mice and Rats,” J PharmPharmacol, 1999, 51(2), 221-226, were more interested in lavender's‘soothing’ properties. They studied the effects of lavender oil on mastcell-mediated allergic reactions in mice and rats and showed that thecomponents of lavender oil inhibit immediate-type allergic reactions byinhibition of mast cell degranulation in vivo and in vitro. Otherresearchers have shown that when lavender is applied topically itstimulates the local circulation (Chevallier, 1996).

[0095] Evening Primrose (oenothera biennis) is a valuable source ofγ-Linolenic Acid (GLA) and other special fatty acids that are essentialfor hormone function, energy flow, cell division, immune responses andmany other aspects of metabolism. These critical fatty acids are used tomake powerful tissue-specific compounds called eicosanoids.

[0096] GLA is effective at very low concentrations (M. S. Manku et al.,Fatty acids in plasma and red cell membranes in normal humans, Lipids,1983, 18(2);906). Doses of 1.4 g/d have resulted in clinically importantreduction of the symptoms of rheumatoid arthritis (L. J. Leventhal etal., “Treatment of Rheumatoid Arthritis with a Gammalinolenic Acid,” AnnInt Med, 1993, 119(9); 867-73). 1-3 g/d is normally recommended fordietary supplements. When tiny amounts of GLA are applied to dry skinthe skin soon shows signs of improvement. This is not surprising sinceone of the early signs of an individual being deficient in GLA is dryskin. Topical GLA application has been shown to promote the healthygrowth of skin, hair, and nails and GLA has been used to successfullytreat skin conditions such as a topic eczema. As well as being good forskin disorders, GLA is reported to be good for arthritis and autoimmuneproblems. (Leung et al., 1996, L. Galland, Increased Requirements forEssential Fatty Acids in Atopic Individuals; Review with ClinicalDescriptions, 1999).

[0097] Individuals with normal metabolism can synthesize GLA from theessential fatty acid linoleic acid. The synthesis is controlled by theenzyme δ-6-desaturase. Interestingly, GLA corrects most of thebiological effects of zinc deficiency (Y. S. Huang et al., “MootBiological Effects of Line Deficiency Corrected by g-Linoleic Acid (18:3omega-6) but not Atherosclerosis, by Linoleic Acid (18:2 omega-6), 1982,41:193-207) indicating that δ-6-desaturase enzyme has a requirement forzinc that is a first-order essential function of zinc. GLA therefore,although not technically a vitamin, is an essential requirement for asignificant number of individuals. Especially those whose δ-6-desaturaseenzyme is blocked or its activity reduced. Interestingly, the ability tosynthesize GLA is affected by factors such as the menstrual cycle anddiet. GLA synthesis is reduced in diabetics or in individuals who fastor consume excessive amounts of carbohydrate (Leung et al., 1996). GLAlevels also decrease with aging.

[0098] GLA can therefore be thought of in similar terms as a vitamin oran essential fatty acid. Moreover, GLA is being extensively studied andhas been shown to be effective in killing cancer cells.

[0099] Eyebright is a plant that has been associated with eye care forgenerations. Eyebright infusions are mildly astringent but gentle enoughto use on eyes. They are stimulating and have antioxidant andanti-inflammatory properties. The plant is recommended both historicallyand in modern literature for treating eye inflammations, particularlyfor conjunctivitis. Eyebright infusions bring rapid relief of redness,swelling and are very good at healing recent eye injuries. They areoften recommended where there is a risk of developing serpiginouscorneal ulcers (Leung et al., 1996).

[0100] Eyebright proves to be most effective when the whole plant isused. The plant is chopped up and applied as a compress (Chevallier,1996). The active biochemicals present in Eyebright plant include thefollowing: Iridoid glycosides, aucubin, catapol and erostoside,eukovoside, geniposide and luproside, gallotanins, caffeic acid andferulic acids. The volatile oil contains trace amounts of the essentialoils as well as beta-sitosterol, oleic acid, palmitic acid and stearicacid. Eyebright also contains miscellaneous unidentified alkaloid, aminoacids, flavonoids and tannins.

[0101] In addition to the water soluble and oil soluble components ofnatural product extracts, in some embodiments the compositions of theinvention may include various bioactive ingredients or cosmeceuticals,including antioxidants, skin whitening agents, elastase inhibitors,vitamins and active agents having anti-inflammatory, antiseptic, orsoothing properties. The composition of the invention may be used inconnection with the treatment of skin disorders, including eczema,psoriasis, acne, photoaging, dermatitis, would healing and dry skin.

[0102] In order to further illustrate the present invention, theexperiments described in the following examples were conducted. Itshould be understood that the invention is not limited to the specificexamples or the details described therein. The results obtained from theexperiments described in the examples are shown in the accompanyingfigures and tables.

EXEMPLARY EMBODIMENTS OF THE INVENTION

[0103] Natural product extracts (both oil soluble and water solubleextracts) and natural product extract compositions containing both oilsoluble and water soluble components were formed according to Examples 1to 26, as shown in Tables 1 to 3 below. TABLE 1 Weight % of Oil SolubleExtracts Silcone Extracts 1 2 3 4 5 6 Mulberry, (Morus alba) dust-powder2.5 grade (China Products) Lavender, (Lavandula angustifolia) fine 2.5chopped plant grade (Aphrodisia) Licorice root, (glycyrrhiza glabra)powder 2.5 grade (Lotus) Arnica (Arnica montana), herb powder 2.5 (SanFrancisco Herb) Eyebright, (euphrasia officinalisa) fine 2.5 choppedplant grade (Aphrodisia) Grape root, (mahonia aquifolium) coarse 2.5chopped grade (Aphrodisia) Silicone fluid DC 345 (Dow Corning) 97.5 97.597.5 97.5 97.5 97.5 Silicone Extracts 1a 2a 3a 4a 5a 6a Green tea(Camelia Sinesis) 2.5 2.5 (China Products) Rosemary powder 2.5(Aphrodisia) Calendula leaves 2.5 Echinacea herb powder (San FranciscoHerb) 2.5 Mulberry, (Morus alba) dust-powder 2.5 grade (China Products)0.65 cst silicone fluid DC200 (Dow 97.5 Corning) Polysynlane (NOF Corp.)97.5 Phenyl Trimethicone (Dow Corning) 97.5 Silicone fluid DC 345 (DowCorning) 97.5 97.5 97.5

[0104] TABLE 2 Extracts Composition - (Parts Per) Glycol Extracts 7 8 910 11 12 13 14 Mulberry, (Morus alba) dust-powder 1 grade (ChinaProducts) Lavender, (Lavandula angustifolia) fine 1 chopped plant grade(Aphrodisia) Licorice root, (glycyrrhiza glabra) 1 powder grade (Lotus)Arnica (Arnica montana), herb powder 1 (San Francisco Herb) Eyebright,(euphrasia officinalisa) fine 1 chopped plant grade (Aphrodisia) Graperoot, (mahonia aquifolium) 1 coarse chopped grade (Aphrodisia) Tea TreeLeaves 1 Evening Primrose Flowers 1 Water 125 125 125 125 125 125 125125 Propylene glycol (Kramer) 10 10 10 10 10 10 4.5 4.5 Method: Mixbotanical and propylene glycol for 5 hrs at room temperature and filterthrough Whatman #1 paper, then add the water at the above ratio and mixto produce the final extract as defined herein and preserve with anappropriate concentration of Germazide ™. Glycol Extracts 7a 8a 9a 10aSea Parsley (Palmaria Palmata) powder 1 Gree Tea (Camelia sinesis)Leaves 1 Rosemary Powder 1 Calendula Leaves 1 Glycerin 3.5 3.5 3.5 Water5.5 5.5 5.5 1-3,Butylene Glycol (Kramer) 9 Method: Mix botanical withpolar solvent for 3 hrs at 40° C. and filter through Whatman #1 paper.An appropriate concentration of Germazide ™ was then added.

[0105] TABLE 3 Compositions Comprising Both Oil Soluble and WaterSoluble Extracts 15 16 17 (Mulberry) Wt. % (Lavender) Wt. % (Licorice)Wt. % Example 1 30 Example 2 30 Example 3 30 Example 7 65 Example 8 65Example 9 65 Additive qs Additive qs Additive qs Lipid & Lipid & Lipid &Preservative Preservative Preservative 18 19 20 (Arnica) Wt. %(Eyebright) Wt. % (Grape Root) Wt. % Example 4 30 Example 5 30 Example 630 Example 10 65 Example 11 65 Example 12 65 Additive qs Additive qsAdditive qs Lipid & Lipid & Lipid & Preservative PreservativePreservative 21 22 23 (Arnica) Wt. % (Eyebright) Wt. % (Grape Root) Wt.% Example 4 30 Example 5 30 Example 6 30 Example 10 65 Example 11 65Example 12 65 Additive qs Additive qs Additive qs Lipid & Lipid & Lipid& Preservative Preservative Preservative 24 25 26 Wt. % Wt. % Wt. % TeaTree Oil 30 Evening 30 Evening 30 Primrose Oil Primrose Oil Example 1350 Example 14 30 Example 14 66.5 Additive qs Additive qs Additive qsLipid & Lipid & Lipid & Preservative Preservative Preservative 27 28 29(Green Tea) Wt. % (Ros. Pwdr) Wt. % (Calendula) Wt. % Example 1a 30Example 2a 30 Example 3a 30 Example 8a 67.5 Example 9a 67.5 Example 10a67.5 Additive qs Additive qs Additive qs Lipid & Lipid & Lipid &Preservative Preservative Preservative

[0106] The term “qs” means a quantity to sufficient to constitute theremaining weight percent of the composition.

[0107] The additive lipid and preservative used in each of Examples15-29 contains 2.35 wt. % phospholipid, 1.6 wt. % Germazide™ MPB, and qswith water.

[0108] Examples 15-29 were formed by mixing the contents of thecomposition, making the contents homogenous by using a Silverson highshear mixer. The composition is then processed through a M110microfluidizer, manufactured by Microfluidics, Inc. of Massachusetts, atapproximately 17,000 psi.

Mulberry Extract

[0109] In Example 1 (Table 1), a hydrophobic solution comprising theorganic oil soluble materials in mulberry root was prepared bycontacting mulberry with a silicone oil and mixing. The resultingcomposition was mixed and filtered to clarity. The Mulberry siliconeextract was examined by HPLC (see FIG. 1) to ensure complete extraction.

[0110] In Example 7, the polar solvent soluble materials were derivedfrom Mulberry. The Mulberry powder extract was obtained by contactingwith propylene glycol and constant stirring for 5 hours at roomtemperature. The resulting composition was diluted with water andGermazide™ MPB was added.

[0111] The HPLC traces for both the polar solvent soluble and apolarsolvent soluble mulberry extracts are shown in FIGS. 1 and 2. It isclear that their overall composition is very different. The chromatogramof the apolar soluble material extracted from mulberry root (FIG. 2) isvery different from the materials found in the chromatogram of the polarsolvent soluble components (FIG. 1).

[0112] The two phases were mixed together along with phospholipid in ahigh shear mixer and then combined using high pressure, high shearprocessing to produce the dispersion of Example 15, containing both thepolar and apolar solvent soluble organic components of mulberry.

[0113] As shown in FIG. 3, the resulting Mulberry extract composition(Example 15) is enriched with powerful tyrosinase inhibitors making itan ideal ingredient in skin whitening products. HPLC chromatograms show,as predicted by the literature, that the dispersion of Example 15contains a vast array of different polar solvent soluble and apolarsolvent soluble compounds. The apolar solvent soluble compounds arebeing effectively extracted into the silicone oil and are thereforepresent in the resulting Mulberry extract composition.

Licorice (glycyrrhiza glabra) Root Extract

[0114] The Licorice extract composition of the invention was preparedfrom two phases; a hydrophobic phase containing the apolar solventsoluble materials in Licorice root and a hydrophilic phase containingthe polar solvent soluble materials.

[0115] In Example 3, a silicone based Licorice extract was prepared.Licorice root powder was contacted with silicone oil and subject tostirring. The resulting composition was filtered to clarity.

[0116] In Example 9, polar solvent soluble materials were obtainedderived from Licorice. Licorice powder was extracted by contactinglicorice with propylene glycol and constant stirring for 5 hours at roomtemperature. The resulting composition was diluted with water andGermazide™ MPB was added.

[0117] The HPLC trace for silicone and aqueous extracts of licorice isshown in FIGS. 4 and 5. Glycyrrhizic acid has been identified and islabeled. FIGS. 4 and 5 show that the Licorice extract composition of theinvention contains a vast array of different compounds. Thechromatograms were run using different wavelengths solvent systems so itis misleading to compare them directly. The size of the peak heights forGylcyrrhizic acid are different because of the different wavelengths.However, the peaks can be clearly seen in both chromatograms.

[0118] The two phases were mixed together along with phospholipid in ahigh shear mixer and then combined them using high pressure, high shearprocessing to produce the dispersion of Example 17, containing both thepolar and apolar solvent soluble organic components of licorice.

[0119]FIGS. 6 and 7 depict the tyrosinase inhibiting activity andmelanocyte pigmentation reduction activity, respectively, of thelicorice extract composition of Example 17. As shown in FIG. 6, licoriceextract contains powerful tyrosinase inhibitors. The effect of theseinhibitors can be demonstrated by either direct tyrosinase inhibition orby measuring the reduction of melanocyte pigmentation. The licoriceextract composition of the present invention is also an antioxidant,making it an ideal ingredient for skin whitening products.

[0120] In addition, a melanocyte pigmentation assay was conducted forthe composition of Example 17. Results of the assay are depicted below.TABLE 4 Melanocyte Pigmentation Reduction of Example 17 Culturepigmentation (OD₄₀₅ nm) % ID conc. mean SE SD CV % inhibition p* Example17 1 0.327 0.004 0.007 2 68 0.00003 Licorice (%) 0.3 0.610 0.015 0.026 441 0.0003 0.1 0.621 0.024 0.042 7 40 0.0005 0.03 0.729 0.017 0.029 4 290.001 0.01 0.842 0.009 0.016 2 18 0.005 untreated — 1.032 0.032 0.055 50 hydro- 30 0.747 0.013 0.023 3 28 0.001 quinone (μM)

[0121] The ID “untreated” refers to a composition which does not containany of the organic components of the licorice extract, and thus is acontrol.

[0122]FIG. 7 depicts the results of the melanocyte pigmentationreduction of Example 17. Example 17 has an EC₅₀ of 0.53% in this assay(see Table 4 and FIG. 7). It is therefore a potent agent for reducingpigmentation and skin whitening.

Lavender (lavandula angustifolia) Extract

[0123] Lavender extract compositions of the invention were obtained.

[0124] In Example 2, a silicone based lavender extract was prepared bycontacting lavender with silicone oil and mixing. The resultingcomposition was filtered to clarity. In Example 8, the polar solventsoluble components of lavender were obtained by contacting lavender withpropylene glycol with constant stirring for 5 hours at room temperature.The resulting composition was diluted with water, and Germazide™ MPB wasadded.

[0125]FIGS. 8 and 9 depict the chromatograms of a water soluble extractand oil soluble extract, respectively, of a lavender extract compositionprepared according to the invention.

[0126] The two phases were mixed together with phospholipid in a highshear mixer and then combined using high pressure, high shear processingto produce the dispersion of Example 16, containing both the polar andapolar solvent soluble organic components of lavender. The dispersion ofExample 16 combines the oil-soluble actives, mainly linalool and linalylacetate, with the polar solvent soluble flavonoids to produce a powerfulcombination that will relax, balance and soothe.

[0127] Elastin is the structural protein that is predominatelyresponsible for the skin's natural elasticity. This elasticity isgradually lost as skin ages or if skin is damaged because elastin isslowly removed through the action of the enzyme elastase. If this enzymeis too active (such as in inflamed skin) the rate of degradation exceedsthe rate of synthesis and the skin quickly looses its suppleness.Cosmetics that inhibit elastase will therefore help improve skin, reducesome of the damaging effects of inflammation and keep skin lookingyounger for longer.

[0128]FIG. 10 shows elastase inhibition by lavender extract andconventional polyethylene glycol (“PG”) extract. Both these extractsinhibit elastase, however the lavender extract composition of theinvention is the better inhibitor. Elastase inhibitors in lavender arewater-soluble and so it was expected that both the conventional PG andthe composition of the invention would show similar inhibition.

[0129] The Example 16 dispersion has the activities necessary tomaintain normal skin and balance combination skin. It is a strongelastase inhibitor, which helps to maintain skin's elasticity andyouthfulness. By inhibiting elastase, the Example 16 dispersion alsoreduces some of the negative effects of inflammation. Theanti-inflammatory properties of Lavender are well supported by publishedarticles. The data described herein demonstrates that Lavender may haveweak anti-inflammatory properties that calm and sooth skin. In addition,cell renewal benefits of lavender are well supported by publishedliterature. The linalool and linalyl acetate seen in the lavenderextract composition of the invention are known to be antiseptic. Theseproperties of Lavender extract make the Example 16 dispersion ideal fornormal and combination skin.

[0130] Table 5 contains the results of the Extra Cellular MatrixDegradation assay. The Example 16 dispersion showed some slightanti-inflammatory activity at the highest concentration tested (3%).These results indicate that the level of a stronger anti-inflammatoryactivity is associated with concentrations of lavender extracts ofgreater than 3%. TABLE 5 Results of the Extra Cellular MatrixDegradation Assay of Example 164 ECM degradation by activated % anti-Conc. neutrophils inflammatory ID (% v/v) mean SE activity Example 16(Lavender) 3 40 1 30 1 58 1 −3 0.3 56 2 2 0.1 54 1 5 0 57 0.5 0Conventional PG 3 54 0 5 Lavender 1 54 1 6 0.3 55 1 3 0.1 54 1 5 0 570.5 0 Assay Controls No neutrophils (spontaneous leeching of 9 0.4 —radiolabeled material from ECM) Neutrophils (degradation of ECM by 27 1— unactivated neutrophils) Activated Neutrophils (degradation of 57 0.5— ECM by neutrophils that have been activated with phorbol ester)Activated Neutrophils + 3% ExCyte ™ 13 1 77 Heather (degradation of ECMby activated neutrophils in presence of ExCyte ™ Heather (positivecontrol for inhibition of matrix degradation))

Arnica Extract

[0131] Arnica extract compositions of the invention were prepared. InExample 4, silicone based arnica extract was prepared by contactingarnica with silicone oil and mixing. The resulting composition wasfiltered to clarity. In Example 10, a polar solvent soluble materials ofarnica were obtained by contacting with propylene glycol with constantstirring for 5 hours at room temperature. The resulting composition wasdiluted with water, and Germazide™ MPB was added. The two phases weremixed together with phospholipid in a high shear mixer and then combinedusing high pressure, high shear processing to produce the dispersion ofExample 18, containing both the polar and apolar solvent soluble organiccomponents of arnica.

[0132] The dispersion of Example 18 was tested for anti-inflammatoryactivity. The results of the testing are depicted in FIG. 11.

Eyebright (euphrasia officinalis)

[0133] An Eyebright extract composition of the invention contains boththe oil-soluble and water-soluble components of the plant, and thus isvery similar to the traditional compresses. The only components missingthat are present in the plant itself are insoluble fibrous materials.The Eyebright extract composition of the invention is therefore moreeffective than conventional Eyebright extracts that normally consist ofeither the water extractable material or the oil extractable material.

[0134] An eyebright extract dispersion was prepared. In Example 5, asilicone eyebright extract was prepared by contacting with silicone oiland mixing. The resulting composition was filtered to clarity. InExample 11, a polar solvent soluble material derived from eyebright wasprepared contacting with propylene glycol with constant stirring for 5hours at room temperature. The resulting composition was diluted withwater, and Germazide™ MPB was added.

[0135]FIGS. 12 and 13 depict the chromatograms of polar solvent soluble(Example 11) and apolar solvent soluble extract (Example 5)compositions.

[0136] The two phases were mixed together with phospholipid in a highshear mixer and then combined using high pressure, high shear processingto produce the dispersion of Example 19, containing both the polar andapolar solvent soluble organic components of eyebright.

[0137]FIG. 14 depicts the results of a cytochrome c reduction assay ofthe eyebright extact composition of the invention, in comparison withSansurf™ oil extracted material, a surfactant-free dispersion oflipophilic materials in water, manufactured by Collaborative Laboratiesof Stony Brook, N.Y.; conventional PG eyebright; and controlcompositions of the composition of the invention and Sansurf™. TheEyebright extract composition of the invention showed significantactivity at the 0.1% concentration. In contrast, the blanks at 0.1% didnot have antioxidant activity. The Eyebright extract composition of theinvention performs better than the other materials tested at allconcentrations up to 0.5%. The apparent activities seen at higherconcentrations are most likely as a result of artifacts such as lightscatter. The Example 19 EC₅₀ Eyebright extract composition of theinvention in the Cytochrome c Reduction assay was 0.07% showing that atthis concentration the composition of the invention is a strongantioxidant.

[0138] Lipid Peroxidation Assay was carried out to alleviate theconcerns raised about the antioxidant activity measured in theCytochrome c reduction assay. It has been suggested that this assay maybe influenced by factors other than oxidation. The results for theExample 19 composition in the Lipid Peroxidation assay are shown inTable 6. TABLE 6 Results of the Lipid Peroxidation Assay of EyebrightCompositions Rate of % OD₅₃₂ at indicated Lipid antioxidant Sample time(min) Peroxidation effect ID conc. % 0 60 120 240 (MOD₅₃₂/min) Example19 1 0.018 0.020 0.021 0.029 46 84 Eyebright 0.2 0.013 0.018 0.028 0.073257 12 0.04 0.014 0.026 0.056 0.088 320 −9 SanSurf ™ Oil 1 0.014 0.0350.060 0.100 360 −23 Extracted 0.2 0.013 0.030 0.062 0.105 396 −35Material 0.04 0.014 0.030 0.059 0.107 395 −35 Conventional 1 0.014 0.0280.058 0.109 410 −40 PG 0.2 0.013 0.031 0.061 0.105 390 −33 Eyebright0.04 0.013 0.034 0.066 0.109 407 −39 Example l9 1 0.020 0.048 0.0740.111 375 −28 Blank 0.2 0.014 0.034 0.062 0.108 397 −35 0.04 0.015 0.0300.059 0.099 359 −23 SanSurf ™ Oil 1 0.020 0.025 0.057 0.105 377 −29Extract 0.2 0.017 0.024 0.056 0.104 381 −30 Material 0.04 0.017 0.0260.055 0.111 408 −39 Blank Vitamin C 0.3 0.011 0.012 0.011 0.013 8 97 0.30.011 0.011 0.011 0.012 3 99 0.03 0.012 0.013 0.016 0.025 54 82 0.030.014 0.0125 0.014 0.0205 30 90 untreated — 0.013 0.013 0.015 0.023 293—

[0139] The blanks listed above contain all the elements of thecomposition without the organic components of the natural products.

[0140] The EC₅₀ for the Eyebright extract dispersion of Example 19 forlipid peroxidation was found to be 0.6%. The Example 19 dispersion istherefore a strong antioxidant while the SanSurf™ Oil ExtractedMaterial, the Conventional PG Eyebright and the blank composition of theinvention did not significantly prevent the production ofmalondialdehyde at any of the concentrations tested and therefore haveno antioxidant properties.

Grape Root (mahonia aquifolium)

[0141] A Grape Root extract composition of the invention was prepared.

[0142] In Example 6, a silicone based Grape Root extract is prepared.Grape Root was extracted by contacting with silicone oil and mixing. Theresulting composition was filtered to clarity. In Example 12, a polarsolvent soluble material derived from Grape Root is prepared. Grape Rootwas extracted by contacting with propylene glycol with constant stirringfor 5 hours at room temperature. The resulting composition was dilutedwith water, and Germazide™ MPB was added. The two phases were mixedtogether with phospholipid in a high shear mixer and then combined usinghigh pressure, high shear processing to produce the dispersion ofExample 20, containing both the polar and apolar solvent soluble organiccomponents of grape root.

[0143]FIG. 15 depicts the results of a cytochrome C reduction assay ofExample 20. These results demonstrate that the Grape Root extractdispersion of the invention (Example 20) is an effective antioxidant atconcentrations of 0.08%.

[0144] The following assays were used for testing the properties of thenatural product extracts of the invention.

Antioxidant Assays Cytochrome C Reduction Assay Materials

[0145] Citrate-phosphate-dextrose solution (“CPD”; C-7165), sodiumchloride (NaCl; S-5886); phosphate-buffered saline (PBS; 1000-3),cytochrome c (C-7752), phorbol 12-myristate 13-acetate (“PMA”; P8139),superoxide dismutase (“SOD”; S-5395) and staurosporine (“STA”; S-4400)were obtained from Sigma. Hank's balanced salt solution (“HBSS”;14025-035) and Lymphoprep™ 1.077 were obtained from Gibco BRL. DextranT-500 (17-0320-01) was obtained from Pharmacia. Wright's stain(LeukoStat™ stain kit) was obtained from Fisher (CS-430). Opticaldensities were determined using a Dynatech MR5000 spectrophotometer.

Methods

[0146] Polymorphonuclear leukocytes (neutrophils or “PMN”) were isolatedusing standard procedures. Blood was drawn from healthy donors usingcitrate-phosphate-dextrose as an anticoagulant. An equal volume of 3%Dextran in 0.9% NaCl was added to settle the majority of erythrocytes.After 20 minutes incubation, the cleared top layer was harvested andcentrifuged at 250 Xg for 10 minutes. The cell pellet was resuspended in0.9% NaCl, layered onto Lymphoprep™ and centrifuged at 400 Xg for 40minutes. The resulting pellet was then subjected to several cycles ofhypotonic lysis (typically 3 cycles) to remove residual erythrocytes.Each lysis cycle involved resuspension of the cell pellet in ice-cold0.2% NaCl for 30 seconds, restoration of isotonicity by the addition ofan equal volume of 1.6% NaCl, followed by centrifugation at 250 g for 10minutes at 4° C. When the erythrocyte removal was complete, the PMN wereresuspended in PBS and stored at 4° C. until required. Purity of thepreparation was checked by staining a cell smear with Wright's stain.

Lipid Peroxidation Assay Materials

[0147] Thiobarbituric acid (“TBA”; T5500), sodium dodecyl sulfate(L4509), butylated hydroxytoluene (B1378), glycine (G7126) andphosphate-buffered saline (“PBS”; 1000-3) were obtained from Sigma.Lecithin (429415) was obtained from Calbiochem. Ethanol (122898) wasobtained from Aaper Alcohol and Chemical Co. Vitamin C (95209) wasobtained from Fluka. Ferric chloride (AR5029) was obtained fromMallinckrodt. Optical densities were determined using a SpectraMax 250spectrophotometer (Molecular Devices). Ultraviolet irradiation wasperformed using a model VWR M-20E Chromato-Vue transilluminator (VWRScientific).

Methods

[0148] The Lipid Peroxidation assay measures inhibition of UV-inducedlipid oxidation. The reaction was performed at room temperature byirradiating liposome solutions, containing varying concentrations oftest samples, with ultraviolet C light.

[0149] The extent of lipid peroxidation induced by the UV light wasdetermined by measuring the consequent production of malondialdehydethat results from breakdown of oxidized lipid. Irradiated liposomesolutions contained 0.8% lecithin and 18% ethanol in PBS, along withtest sample or control. Vitamin C, a peroxyl radical scavenger, wasincluded in each assay as a control for inhibition of lipid oxidation.Duplicate aliquots of the irradiation mixtures were harvested at severaltime points and their malondialdehyde contents were measured using the“thiobarbituric acid reaction”. TBA reaction mixtures contained 300 μMbutylated hydroxytoluene, 300 μM FeCl₃, 16 mM TBA, 0.14% sodium dodecylsulfate, 90 μM glycine (pH 3.6) and 3%(v/v) irradiation mixture aliquot.The mixtures were heated at 100° C. for 15 minutes, cooled to roomtemperature and their absorbencies were read at 532 and 650 nm. Thereadings at 650 nm were subtracted from those at 532 nm to correct forturbidity. These corrected absorbencies were plotted as a function ofirradiation time and the rate of lipid peroxidation was determined bylinear regression curve fit. An EC₅₀ (the concentration of test samplethat inhibits 50% of the rate of lipid peroxidation) was calculated foreach sample.

Anti-Inflammation Assay Extra Cellular Matrix Degradation (ECM) AssayMaterials

[0150] Ammonium hydroxide (A-6899), streptomycin sulfate (S-0890)citrate-phosphate-dextrose solution (“CPD”; C-7165), sodium chloride(NaCl; S-5886); phosphate-buffered saline (PBS; 1000-3), phorbol12-myristate 13-acetate (“PMA”; P8139), and sodium azide (S-8032) wereobtained from Sigma. Minimal Essential Medium (“MEM”; 11095-072),tryptose phosphate (18050-013) heat-inactivated fetal bovine serum(“FBS”; 10082-147) Hank's balanced salt solution (“HBSS”; 14025-035) andLymphoprep™ 1.077 were obtained from Gibco BRL. R-22 cells were obtainedfrom Dr. S. Simon (Dept. of Pathology, SUNY Stony Brook). Dextran T-500(17-0320-01) was obtained from Pharmacia. Wright's stain (LeukoStat™stain kit) was obtained from Fisher (CS-430). ExCyte™ Heather wasobtained from Collaborative Laboratories.

[0151] For the production of radiolabeled ECM plates, R-22 smooth musclecells were seeded into 24-well plates and grown in maintenance medium(MEM containing 10% FBS, 2% tryptose phosphate, 100μg/ml streptomycin).Upon reaching confluence, the cells were switched into labeling medium(maintenance medium supplemented with 50 mg/ml ascorbic acid and 0.5μCi/ml L-[2,3,4,5-³H]-proline) and maintained in this medium for 2weeks. The plates were then harvested by aspirating the labeling mediumand lysing the cells by 5 minute incubation in 25 mM ammonium hydroxidefollowed by 3 cycles of 5 minute incubation in sterile deionized water.Prior to use, the plates were stored at 4° C. with 50 μl/well of 0.02%sodium azide.

[0152] Polymorphonuclear leukocytes (neutrophils or “PMN”) were isolatedusing standard procedures. Blood was drawn from healthy donors usingcitrate-phosphate-dextrose as anticoagulant. An equal volume of 3%Dextran in 0.9% NaCl was added to settle the majority of erythrocytes.After 20 minutes incubation, the cleared top layer was harvested andcentrifuged at 250 Xg for 10 minutes. The cell pellet was resuspended in0.9% NaCl, layered onto Lymphoprep™ and centrifuged at 400 Xg for 40minutes. The resulting pellet was then subjected to several cycles ofhypotonic lysis (typically 3 cycles) to remove residual erythrocytes.Each lysis cycle involved resuspending the cell pellet in ice-cold 0.2%NaCl for 30 seconds, restoration of isotonicity by addition of an equalvolume of 1.6% NaCl, followed by centrifugation at 250 g for 10 minutesat 4° C. When erythrocyte removal was complete, the PMN were resuspendedin PBS and stored at 4° C. until use. Purity of the preparation waschecked by staining a cell smear with Wright's stain.

Methods

[0153] For the ECM degradation assay, radiolabeled ECM plates werewashed 3 times with HBSS to remove azide. To start the degradationreaction, 1 ml of HBSS containing 5 nM PMA, 1×10⁶ PMN, and the indicatedtest sample concentration was added to 3 wells of an ECM plate. In eachassay, there were also 4 control conditions (triplicate wells for each).These were HBSS alone (to correct for leeching of unincorporatedradiolabel), cells in HBSS (to monitor degradation by “unstimulated”PMN), cells in HBSS with 5 nM PMA (maximal stimulated matrixdegradation), and cells in HBSS with 5 nM PMA and 3% ExCyte™ Heather(positive control for inhibition of matrix degradation), available fromCollaborative Laboratories. Upon addition of reaction mixtures, theplates were incubated at 37° C. for 4 hours. ECM degradation in eachwell was scored by scintillation counting to measure radioactivityreleased into the supernatant as well as that remaining in the residualmatrix. Using these 2 measures, the % ECM degradation was calculated foreach well. An EC₅₀, the concentration of test sample that decreased ECMdegradation by 50% relative to the maximal stimulated matrix degradationcondition, was calculated where possible.

Elastase Inhibition Assay Materials

[0154] Tris[hydroxymethyl]aminomethane (“Tris”; T-1410), sodium chloride(NaCl; S-5886), dimethyl sulfoxide (“DMSO”; D-8779), sodium acetate(S-8625) and hydrochloric acid (“HCl”; H-7020) were obtained from Sigma.Human neutrophil elastase (“HNE”; 16-14-051200) was obtained from AthensResearch. MethoxysuccinylAAPVpNA (“peptide”; L-1335) was obtained fromBachem. Optical densities were determined using a Dynatech MR5000spectrophotometer.

Methods

[0155] The assay was performed in a 96-well plate with triplicate wellsfor each reaction condition. Reaction mixtures contained 63 mM Tris-HCl(pH 8.0), 195 mM NaCl, 5 mM sodium acetate, 1.5% DMSO, 300 μg/μlpeptide, 1.5 μg/μl HNE and test sample as indicated. The reaction wasstarted by addition of HNE and followed by measuring A₄₀₅ at minuteintervals for 10 minutes. The reaction rate was determined from theslope of a straight line fitted to the data plot.

Inhibition of UV-Induced PGE₂ Production

[0156] This assay measures production of PGE₂ by keratinocytes exposedto UVB radiation.

Materials

[0157] HaCaT cells, a spontaneously immortalized human keratinocyte line(Boukamp et al, J Cell Biology 106 (1988) 761-771), were obtained fromDr. Norbert Fusenig (German Cancer Research Center).Tris[hydroxymethyl]aminomethane (T-1410), sodium chloride (S-5886),ethylenediaminetetraacetic acid (E-4884), and phosphate-buffered saline(“PBS”; 1000-3), Neutral Red (N-6634) and glucose (G-5400) were obtainedfrom Sigma. Ethanol (A405P-4) was obtained from Fisher Scientific.Acetic acid (AC110) was obtained from Spectrum Chemical Corp. Dulbecco'sModified Eagle's Medium (“DMEM”; 11885-076), heat-inactivated fetalbovine serum (“FBS”; 10082-147), and 5000 units/ml penicillin/5000 μg/μlstreptomycin (15070-063) were obtained from Gibco BRL. PGE₂ EIA kits(DE0100) were obtained from R&D Systems. UBL model FSX24T12/UVB-HO bulbs(National Biological Corp.) were used for cell irradiation. Bulb outputwas measured with an International Light Model IL1700 radiometer.Absorbances were measured using a Dynatech MR5000 spectrophotometer.

Methods

[0158] Cells were seeded into 12-well plates in DMEM containing 10% FBS,50 units/ml penicillin and 50 μg/μl streptomycin (“medium”). When thecells were approximately 65% confluent they were switched into mediumcontaining varying concentrations of test samples or indomethacin(positive control for inhibition of PGE₂ production). After a 12-hourpreincubation, the media were removed and the cells were washed with,and transferred into PBS-glucose (PBS containing 5.5 mM glucose). Theywere immediately irradiated with 11 mJ/cm² of UVB (approx. 45 secondexposure) and transferred back into media containing the sameconcentrations of test samples or controls as for the preincubationperiod. After a further 8 hour incubation, the media were collected andstored at −70° C. After removal of the media, the cells were fed withmedium containing Neutral Red dye and incubated at 37° C. for 3 hours.The cells were then washed with buffer and internalized dye wasextracted with an ethanolacetic acid solution. Extracted Neutral Red wasdetermined by measuring its absorbance at 550 nm. The levels of PGE₂ inthe supernatants were measured using a commercially available EIA kit.

Skin Whitening Assays Tyrosinase Inhibition Activity Materials

[0159] The following materials were used in the tyrosinaseassay:—Tyrosine T8909, Tyrosinase (mushroom) T7755, Hydroquinone H9003,and Sodium Phosphate S7907 and S8282 were obtained from Sigma. Opticaldensity readings were determined using a Dynatech MR5000spectrophotometer.

Methods

[0160] Tyrosinase activity was determined by measuring the rate ofchange of optical density at 490 nm as tyrosine was converted intodopachrome. (Tyrosinase catalyzes the conversion of tyrosine into DOPAquinone, which spontaneously converts into dopachrome.) The rate oftyrosinase activity in the presence of various concentrations of testsample was measured at room temperature in a reaction mixture containing50 mM sodium phosphate (pH 6.75), 275 μM tyrosine and 25 U/ml mushroomtyrosinase. Readings were made at one minute intervals for ten minutesand the reaction rate was calculated by linear regression. Eachconcentration was run in triplicate. Inhibition of tyrosinase activitywas expressed as a percentage of the activity measured for the untreatedcontrol mixture (no test sample). An EC₅₀ (the concentration of testsample that inhibits 50% of tyrosinase activity) was calculated.Hydroquinone was tested in each assay as a positive control forinhibition of tyrosinase activity.

Melanocyte Pigmentation Assay Materials

[0161] The following materials were used in the melanocyte pigmentationassay. Cloudman S91 cells (36-1-38C8-16) were obtained from AmericanType Culture Collection. Dulbecco's Modified Eagle's Medium (“DMEM”;11885-076), heat-inactivated horse serum (26050-088), heat-inactivatedfetal bovine serum (10082-147) and 5000 units/ml penicillin/50 μg/mlstreptomycin (15070-063) were obtained from Gibco BRL.Phosphate-buffered saline (PBS; 1000-3), dimethyl sulfoxide (D2650),hydroquinone (H9003), α-melanocyte stimulating hormone (M4135),trichloroacetic acid (T9159) and sodium hydroxide (S8045) were obtainedfrom Sigma. Optical densities were determined using a Dynatech MR5000spectrophotometer.

Methods

[0162] The melanocyte pigmentation assay measures a test sample'sability to inhibit pigmentation. Cloudman S91 melanocytes were seededinto multiwell plates in medium (Dulbecco's Modified Eagle's Mediumcontaining 15% heat-inactivated horse serum, 2.5% heat-inactivated fetalbovine serum, 50 units/ml penicillin and 50 μg/ml streptomycin)containing 10 nM ∝-melanocyte stimulating hormone and varyingconcentrations of the test sample or 30 μM hydroquinone (the positivecontrol for inhibition of pigmentation). After a 5-day treatment periodwith each condition tested in triplicate wells, the cells were harvestedby washing with PBS and adding 5% trichloroacetic acid to each well.After a 15 minute incubation the acid was aspirated and replaced with200 μl/well of 10% dimethyl sulfoxide/1N sodium hydroxide. The plateswere sealed and incubated at 65° C. for 30 minutes. 200 μl of eachdigestion mixture was then transferred to a well of a 96-well microtiterplate and optical densities were read at 405 nm.

[0163] Inhibition of pigmentation is expressed as the percentagedecrease in optical density compared to that for untreated cells (notest sample or hydroquinone). An EC₅₀ (the concentration of test samplethat inhibits 50% of pigmentation) was calculated for each sample.

What is claimed
 1. A stable, homogeneous dispersion comprising a watersoluble natural product extract and an oil soluble natural productextract, wherein said composition does not comprise a surface activeagent.
 2. The composition of claim 1 wherein said composition comprisesa non-surface active lipid phosphate.
 3. A method of forming a stable,homogeneous dispersion comprising water soluble components of a naturalproduct extract and oil soluble components of a natural product extract,said composition formed by the steps of: obtaining a first naturalproduct extract comprising a polar solvent and water based components ofa natural product; obtaining a second natural product extract comprisingan apolar solvent and oil soluble components of a natural product;combining said first extract and said second extract to form a firstcomposition; adding about 0.01 to 8% by weight of a non-surface activelipid phosphate or a surface active agent; and subjecting said mixtureto high pressure high shear processing to form a stable, homogeneousdispersion.
 4. The method of claim 3 wherein said step of subjectingsaid composition to high pressure high shear processing occurs at apressure of about 11,000 to about 27,000 psi and a shear rate which issufficient to form a dispersion having an average particle size of fromabout 200 to about 1,000 nm.
 5. The dispersion of claim 3, wherein saidpolar solvent is selected from the group consisting of water; a mono,di, tri or polyhydroxy alkyl derivative; a mono, di, tri orpolyhalogenated alkyl derivative; a mono, di, tri or poly alkyl etherderivative; or a mono, di, tri or poly carboxy alkyl derivative, ormixtures thereof.
 6. The method of claim 3, wherein said apolar solventis selected from the group consisting of a mono, di, tri or polyalkylester or ether of a mono, di, tri or polyhydroxy compound; a saturated,unsaturated, linear, branched, or cyclic hydrocarbon; a saturated,unsaturated, linear or branched C₈ to C₃₀ fatty acid; a branched,linear, or cyclical silicone or silicone derivative; or a homopolymer orheteropolymer fluid formed by the polymerization of alkylene oxidemonomers.
 7. The method of claim 6, wherein said apolar solvent isselected from the group consisting of vegetable oil, soybean oil, babasuoil, castor oil, cottonseed oil, grapeseed oil, rice bran oil, canolaoil, palm oil, palm kernel oil, olive oil, linseed oil, coconut oil,sunflower oil, safflower oil, peanut oil, corn oil, mineral oil,petrolatum, hydrogenated polyisobutene, permethyl fluids, polyisobutene,polybutene, cyclomethicone, dimethicone polysiloxane, dimethicinol,polysiloxanes, polyalkyl siloxanes, polyarylsiloxanes, polyalkylarylsiloxanes, polysiloxane copolymers, polypropylene oxide, polybutyleneoxide, isopropyl palmitate, diisopropyl adipate or mixtures thereof. 8.The method of claim 3, wherein said first composition further comprisesa non-surface active lipid phosphate.
 9. The method of claim 3, whereinsaid polar solvent is a mixture of water and propylene glycol.
 10. Themethod of claim 3, wherein said first and second natural products arethe same product.
 11. The method of claim 3, wherein said first andsecond natural products are extracted from different natural products.12. The method of claim 3, wherein said first and second naturalproducts are extracted from a mixture of different natural products. 13.The method of claim 3, wherein said first and second natural productsare extracted from a mixture of different natural products,respectively.
 14. The method of claim 3, wherein said natural product isselected from the group consisting of mulberry, lavender, licorice root,arnica, eyebright and grape root.